Bipolar mitotic spindle assembly is critically important for proper segregation of a duplicated genome. Most spindle components have now been identified, but the ways they self-organize and generate and respond to physical forces remain largely unexplored topics in basic biomedical research. This gap in our understanding of spindle assembly mechanics persists despite years of research in this area because very few studies have provided quantitative, systems-level information about the spindle or the forces it can produce. To address this gap we have proposed an integrated set of experimental approaches to investigate molecular and mechanical aspects of spindle assembly, with a focus on the microtubule-based motor cytoplasmic dynein. This motor plays critically important roles in determining spindle shape, but its multifunctional character, large size and structural complexity have made it a difficult subject to study, providing a small frontier in the otherwise well-explore field of mitosis. Based on preliminary data, we hypothesize that cell cycle-dependent interactions with other proteins regulate dynein function, conferring a specific ability to crosslik and slide antiparallel microtubules. By characterizing the composition of the responsible dynein-containing motor complex and the forces it generates, we hope to elucidate a mitotic function of dynein and, therefore, one that can be selectively targeted in dividing cells. To test our central hypothesis we propose three aims. The first uses a proteomics based screen to identify proteins whose interactions with dynein are either cell-cycle dependent or regulated by mitotic signaling pathways. In the second aim, microneedle-based force measurements will be used to quantify dynein-dependent forces generated during spindle assembly. Lastly, the third aim describes the design and calibration of a novel, genetically encoded force-probe for high-resolution mapping of sliding-filament forces within the spindle. Completion of the work proposed in these aims is expected to produce a fundamental advance in our basic understanding of dynein function and to identify molecular targets for the eventual development of new anti-cancer drugs. It will also provide much needed systems-level characterization of integrated spindle forces as well as quantitative information needed to resolve conflicting models of emergent spindle properties like bipolarity and length. In addition, due to their potential for broad application, development of th new methods and approaches proposed herein will greatly expand future studies aimed at characterizing mechanical forces and force- initiated signaling within cells.